Epithelial-mesenchymal plasticity determines estrogen receptor positive breast cancer dormancy and epithelial reconversion drives recurrence

More than 70% of human breast cancers (BCs) are estrogen receptor α-positive (ER+). A clinical challenge of ER+ BC is that they can recur decades after initial treatments. Mechanisms governing latent disease remain elusive due to lack of adequate in vivo models. We compare intraductal xenografts of ER+ and triple-negative (TN) BC cells and demonstrate that disseminated TNBC cells proliferate similarly as TNBC cells at the primary site whereas disseminated ER+ BC cells proliferate slower, they decrease CDH1 and increase ZEB1,2 expressions, and exhibit characteristics of epithelial-mesenchymal plasticity (EMP) and dormancy. Forced E-cadherin expression overcomes ER+ BC dormancy. Cytokine signalings are enriched in more active versus inactive disseminated tumour cells, suggesting microenvironmental triggers for awakening. We conclude that intraductal xenografts model ER + BC dormancy and reveal that EMP is essential for the generation of a dormant cell state and that targeting exit from EMP has therapeutic potential.

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April 2020
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Life sciences study design
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Sample size
Sample size was estimated based on previous experience with the experimental approaches (Sflomos, G. et al., Cancer Cell, 2016). For growth analyses, at least 4 mice with at least 2 tumors/mouse are required (total of 8 tumors). For metastasis analyses, at least 5 mice are required.
Data exclusions For the scRNAseq, cells with less than 500 genes and/or higher than 20% mitochondrial reads were removed from the analysis. For qPCR, replicates in which the CT value is higher than 35 (not detected) were excluded. For growth and metastasis analysis, outliers were removed by calculating the the upper and lower boundary, any value that is 1.5 x Interquartile (IQR) greater than the third quartile was designated as an outlier, and any value that is 1.5 x IQR less than the first quartile was also designated as an outlier. No data were excluded from Immunofluorescence and stereoscope analysis, mammary gland weight, mouse body weight, proliferation analysis, western blot, and collagen quantification.

Replication
All experiments were repeated at least 3 independent times at the exception of the Western blot in Extended Data Figure 7h and 7i, which was used to validate successful overexpression. Replication was successful.
Randomization Before initiation of treatment (DOX), we ensured that control and treatment cohorts have similar primary tumor radiance values. For the rest of the experiments, no randomization was needed.

Blinding
Image analysis in Figure 2g-n, 4g,m, Figure 3b-d, and Figure 6k-o was carried out by "blinded" experimenter i.e. the experimenter did not know the corresponding genotype. For the rest of the experiments, investigators were not blinded to group allocation. For growth assays and metastasis analysis, no blinding was applied as mice are separated in different cages and labeled with their respective condition. For the rest of the experiments, blinding was not necessary because the readout was automated i.e. qPCR, or run through R i.e. scRNAseq, velocity analysis and the experimenter needs to know the corresponding genotypes/conditions.

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Laboratory animals
All mice were maintained and handled according to Swiss guidelines for animal safety with a 12-h-light-12-h-dark cycle, controlled temperature (22 ± 2 degrees Celsius) and humidity (55% ± 10%) , and food and water ad libitum. Experiments were performed in accordance with protocol VD1865.5 approved by the Service de la Consommation et des Affaires Vétérinaires, Canton de Vaud, Switzerland. NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) and NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CAG-EGFP)1Osb/SzJ (NSG-EGFP) mice were purchased from Charles River and The Jackson Laboratory. 8-16-week-old NSG or NSG-EGF female mice were used in this study.

Wild animals
No wild animals were used in this study.
Field-collected samples No field-collected samples were used in this study.

Ethics oversight
All mice were maintained and handled according to Swiss guidelines for animal safety and experiments were performed in accordance with protocol VD1865.5 approved by Service de la Consommation et des Affaires Vétérinaires, Canton de Vaud, Switzerland.
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